Amplification techniques for the detection of target nucleic acids in biological samples offer high sensitivity and specificity for the detection of infectious organisms and genetic defects. Copies of specific sequences of nucleic acids are synthesized at an exponential rate through an amplification process. Examples of these techniques are the polymerase chain reaction (PCR), disclosed in U.S. Pat. Nos. 4,683,202 and 4,683,195 (Mullis); the ligase chain reaction (LCR) disclosed in EP-A-320 308 (Backman et al); and gap filling LCR (GLCR) or variations thereof, which are disclosed in WO 90/01069 (Segev), EP-A-439-182 (Backman, et al), GB 2,225,112A (Newton, et al) and WO 93/00447 (Birkenmeyer et al.). Other amplification techniques include Q-Beta Replicase, as described in the literature; Strand Displacement Amplification (SDA) as described in EP-A-497 272 (Walker), EP-A-500 224 (Walker, et al) and in Walker, et al., in Proc. Nat. Acad. Sci. U.S.A., 89:392 (1992); Self-Sustained Sequence Replication (3SR) as described by Fahy, et al. in PCR Methods and Applications 1:25 (1991); and Nucleic Acid Sequence-Based Amplification (NASBA) as described in the literature.
These reactions, particularly where requiring thermal cycling, are usually carried out in microfuge-type tubes such as the SlickSeal.TM. tubes available from National Scientific (San Rafael, Calif.), or in Thin-Walled GeneAmp.TM. tubes available from Perkin-Elmer (Norwalk, Conn.). Another type of reaction container is a strip of microfuge reaction vessels combined with a strip of domed caps as described in EP-A-488 769 and marketed by Perkin-Elmer (Norwalk, Conn.) as MicroAmp.TM. for use with a Perkin-Elmer 9600 thermal cycler. In a typical procedure, after performing the amplification reaction the tubes are opened and a portion of the amplified reaction product is transferred to a detection apparatus such as a microtiter plate, a gel or other detection apparatus.
A major problem with such nucleic acid amplification procedures is the contamination risk when the amplification vessels are opened up. Spillage, droplet formation and/or aerosols can be generated when the caps are removed in order to remove a portion of the amplified reaction product for detection analysis. This can spread the amplified product throughout the lab by airborne droplets or on equipment and can contaminate un-amplified samples and/or reagents. This will quickly lead to false positive results. Extreme precautions must be taken to prevent such contamination. Physical separation between sample preparation, amplification and detection areas has been customarily used in the art. It is quite cumbersome, expensive and requires rigorous training to prevent transfer of lab coats, gloves, pipettes or laboratory equipment between such segregated areas.
U.S. Pat. No. 5,229,297 and corresponding EP-A- 0 381 501 (Kodak) disclose a cuvette for carrying out amplification and detection of nucleic acid material in a closed environment to reduce the risk of contamination. The cuvette is a closed device having compartments that are interconnected by a series of passageways. Some of the compartments are reaction compartments for amplifying DNA strands, and some of the compartments are detection compartments having a detection site for detecting amplified DNA. Storage compartments may also be provided for holding reagents. Samples of nucleic acid materials, along with reagents from the storage compartments, are loaded into the reaction compartments via the passageways. The passageways leading from the storage compartment are provided with one-way check valves to prevent amplified products from back-flowing into the storage compartment. The sample is amplified in the reaction compartment, and the amplified products are transferred through the interconnecting passageways to detection sites in the detection compartment by applying external pressure to the flexible compartment walls to squeeze the amplified product from the reaction compartments through the passageways and into the detection compartments. Alternatively, the cuvette may be provided with a piston arrangement to pump reagents and/or amplified products from the reaction compartments to the detection compartment.
Although the cuvette disclosed in EP 0 381 501 A2 (Kodak) provides a closed reaction and detection environment, it has several significant shortcomings. For example, the multiple compartments, multiple passageways, check valves and pumping mechanisms present a relatively complicated structure that requires much effort to manufacture. Also, the shape and configuration of the cuvette disclosed in EP 0 381 501 A2 do not allow it to be readily inserted into conventional thermal cycling devices. In addition, the fluid transfer methods utilized by the cuvette call for a mechanical external pressure source, such as a roller device applied to flexible side walls or the displacement of small pistons. Conventional thermal cycling devices are not readily adapted to include such external pressure sources, and mechanical pressure applied to the flexible walls can rupture these walls, especially if the cuvette is misaligned. Rupture of the flexible wall of an external compartment containing the amplified reaction product would lead to contamination of the inside of the instrument and possibly the entire laboratory. Finally, the apparatus described in this reference is quite limited in terms of throughput of the disclosed devices. The system does not provide the desired flexibility for manufacturing.
French patent publication No. FR 2 672 301 (to Larzul) discloses a similar hermetically closed test device for amplification of DNA. It also has multiple compartments and passages through which sample and/or reagents are transferred. The motive forces for fluid transport are described as hydraulic, magnetic displacement, passive capillarity, thermal gradient, peristaltic pump and mechanically induced pressure differential (e.g. squeezing).
Other methods applied in the art to deal with contamination issues are chemical in nature. One such method is described in U.S. Pat. No. 5,035,996 (Hartley, Life Technologies, Inc). It involves incorporating into the amplification product a ribonucleoside triphosphate (rNTP) or deoxyribonucleoside triphosphate (dNTP) base that is not generally found in the sample to be analyzed: for example dUTP in the case of DNA analysis. The amplified product will thus have a sequence that has Uracil in multiple positions. The enzyme uracil DNA glycosylase (UDG) is added to samples prior to amplification. This will cause digestion of any contaminating reaction product (containing Uracil) without affecting the natural DNA in the sample.
This method will work for PCR but has limited potential for LCR. It can not be applied to blunt end LCR, and has a very limited potential for gap LCR. In gap LCR, it is not practical to incorporate more than a few uracil bases to fill the gap. Action of UDG will be at one site only, as opposed to a large number of sites in PCR amplification. Although this method has been commercialized by Roche Diagnostics as a way of inactivation of Amplicor.TM. DNA amplification assays, it cannot be applied to a variety of amplification reactions.
Other methods used to minimize the risk of contamination include the destruction of the amplified reaction product as well as any polynucleotide reagents after completion of the detection reaction. Such a method has been described by Celebuski in co-owned U.S. patent application Ser. No. 07/863,622, entitled "Methods for Inactivating Nucleotide Sequences and Metal Chelates for use Therein", filed Apr. 3, 1992. The inactivation method utilizes a divalent metal chelate such as copper phenanthroline complex and a dilute solution of hydrogen peroxide added to the reaction products and optionally to all equipment. This composition is very effective at cleaving all DNA into small fragments that are incapable of amplification. Accordingly, it is used after detection of amplification product, rather than prior to amplification.
Chemical measures such as UDG and metal chelates are effective in preventing minor contamination, but are less satisfactory in the case of major contamination involving droplets of reaction product. Thus the need to perform the amplification reaction in a closed system has been realized in the art in such documents as EP 0 381 501 A2, EP 0 550 090 A1 and U.S. Pat. No. 5,229,297. These documents describe such closed-reaction disposables.
Each of the patents, patent applications and literature documents specifically cited above or below is incorporated herein in its entirety by reference.
With these limitations of prior art, it is thus an important object of the invention to seek amplification reaction vessels and methods of use that will minimize contamination risk. A further object is to provide a disposable reaction vessel and method whereby an amplified reaction sample can be removed without removing a sealing cap; since cap removal tends to spread aerosol contamination. A further object of the invention is to provide a sealed disposable reaction vessel and method whereby an amplified reaction sample can be withdrawn with minimal disturbance to the seal of the vessel.
Another object of the invention is to provide a formulation that is suitable for unit dose preparation of reaction vessels such as the one described herein.
Yet another object of the invention is to provide a reaction vessel that is at once compatible with commercial thermal cyclers, for example the Perkin-Elmer 480, as well as with automated detection instrumentation such as those utilizing Microparticle Enzyme ImmunoAssay (MEIA) technology.
These and other objectives are met in the present invention as described below.